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1.
PLoS One ; 15(8): e0236781, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32776961

RESUMO

It has been reported that Bach1-deficient mice show reduced tissue injuries in diverse disease models due to increased expression of heme oxygenase-1 (HO-1)that possesses an antioxidant function. In contrast, we found that Bach1 deficiency in mice exacerbated skeletal muscle injury induced by cardiotoxin. Inhibition of Bach1 expression in C2C12 myoblast cells using RNA interference resulted in reduced proliferation, myotube formation, and myogenin expression compared with control cells. While the expression of HO-1 was increased by Bach1 silencing in C2C12 cells, the reduced myotube formation was not rescued by HO-1 inhibition. Up-regulations of Smad2, Smad3 and FoxO1, known inhibitors of muscle cell differentiation, were observed in Bach1-deficient mice and Bach1-silenced C2C12 cells. Therefore, Bach1 may promote regeneration of muscle by increasing proliferation and differentiation of myoblasts.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Diferenciação Celular , Músculo Esquelético/fisiologia , Mioblastos/citologia , Regeneração , Proteínas Smad/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Inativação Gênica , Camundongos , Músculo Esquelético/citologia , Transcriptoma/genética
2.
Oncotarget ; 9(36): 24291-24303, 2018 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-29849941

RESUMO

The aim of this study was to identify novel liver metastasis-correlated proteins of PanNEN by proteomics to compare pancreatic tumor (PT) with paired metastatic liver tumor (LT). Of 118 surgical cases with PanNEN, 7 cases with formalin-fixed paraffin-embedded (FFPE) tissues of both PT and paired LT were evaluated by proteomics. Tumor cells were selectively collected from FFPE tissues by laser capture microdissection. A total of 3,722 proteins were detected from extracted peptides by mass spectrometry-based shotgun analysis. Selection of the candidate proteins expressed differently between PT and LT were performed by semi-quantitative comparison in silico and confirmation with immunohistochemistry. We focused on ANXA6, CNPY2, RAB11B and TUBB3, all of which had higher expressions in LT. In all surgical cases with FFPE samples, liver recurrence-free survival (RFS) was evaluated in correlation to the expression of the candidate proteins in PT by immunohistochemistry. Liver RFS was significantly poorer in CNPY2 positive patients than in negative patients (10-year liver RFS; 39.8% vs. 92.3%, p = 0.012). Also, liver RFS tended to be poorer in ANXA6 positive patients than in those who were negative (10-year liver RFS; 51.4% vs. 95.0%, p = 0.099). In the multivariate analysis, the independent predictors of liver RFS were CNPY2 positivity (HR: 6.19, 95 % CI: 1.47-42.79, p = 0.011) and tumor size ≥ 42 mm (HR: 4.63, 95 % CI: 1.03-23.23, p = 0.045). In conclusion, CNPY2 is a novel liver metastasis-correlated protein of PanNEN.

3.
Genes Cells ; 23(6): 406-417, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29701317

RESUMO

Population-based prospective cohort studies are indispensable for modern medical research as they provide important knowledge on the influences of many kinds of genetic and environmental factors on the cause of disease. Although traditional cohort studies are mainly conducted using questionnaires and physical examinations, modern cohort studies incorporate omics and genomic approaches to obtain comprehensive physical information, including genetic information. Here, we report the design and midterm results of multi-omics analysis on population-based prospective cohort studies from the Tohoku Medical Megabank (TMM) Project. We have incorporated genomic and metabolomic studies in the TMM cohort study as both metabolome and genome analyses are suitable for high-throughput analysis of large-scale cohort samples. Moreover, an association study between the metabolome and genome show that metabolites are an important intermediate phenotype connecting genetic and lifestyle factors to physical and pathologic phenotypes. We apply our metabolome and genome analyses to large-scale cohort samples in the following studies.


Assuntos
Genoma Humano , Genômica/métodos , Metabolômica/métodos , Humanos , Fenótipo , Estudos Prospectivos
4.
Proteomics ; 17(13-14)2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28556466

RESUMO

Ultimately, cell biology seeks to define molecular mechanisms underlying cellular functions. However, heterogeneity within cell populations must be considered for optimal assay design and data interpretation. Although single-cell analyses are desirable for addressing this issue, practical considerations, including assay sensitivity, limit their broad application. Therefore, omics studies on small numbers of cells in defined subpopulations represent a viable alternative for elucidating cell functions at the molecular level. MS-based proteomics allows in-depth proteome exploration, although analyses of small numbers of cells have not been pursued due to loss during the multistep procedure involved. Thus, optimization of the proteomics workflow to facilitate the analysis of rare cells would be useful. Here, we report a microproteomics workflow for limited numbers of immune cells using non-damaging, microfluidic chip-based cell sorting and MS-based proteomics. Samples of 1000 or 100 THP-1 cells were sorted, and after enzymatic digestion, peptide mixtures were subjected to nano-LC-MS analysis. We achieved reasonable proteome coverage from as few as 100-sorted cells, and the data obtained from 1000-sorted cells were as comprehensive as those obtained using 1 µg of whole cell lysate. With further refinement, our approach could be useful for studying cell subpopulations or limited samples, such as clinical specimens.


Assuntos
Separação Celular/métodos , Espectrometria de Massas/métodos , Microfluídica/métodos , Proteoma/metabolismo , Humanos , Microfluídica/instrumentação , Proteoma/análise , Células THP-1
5.
Front Genet ; 8: 17, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28270832

RESUMO

Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex.

6.
J Biol Chem ; 292(19): 8019-8037, 2017 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-28302717

RESUMO

The capacity of the liver to regenerate is likely to be encoded as a plasticity of molecular networks within the liver. By applying a combination of comprehensive analyses of the epigenome, transcriptome, and proteome, we herein depict the molecular landscape of liver regeneration. We demonstrated that histone H3 Lys-4 was trimethylated at the promoter regions of many loci, among which only a fraction, including cell-cycle-related genes, were transcriptionally up-regulated. A cistrome analysis guided by the histone methylation patterns and the transcriptome identified FOXM1 as the key transcription factor promoting liver regeneration, which was confirmed in vitro using a hepatocarcinoma cell line. The promoter regions of cell-cycle-related genes and Foxm1 acquired higher levels of trimethylated histone H3 Lys-4, suggesting that epigenetic regulations of these key regulatory genes define quiescence and regeneration of the liver cells. A quantitative proteome analysis of the regenerating liver revealed that conditional protein degradation also mediated regeneration-specific protein expression. These sets of informational resources should be useful for further investigations of liver regeneration.


Assuntos
Regulação da Expressão Gênica , Histonas/química , Regeneração Hepática , Fígado/metabolismo , RNA Mensageiro/metabolismo , Motivos de Aminoácidos , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Epigênese Genética , Proteína Forkhead Box M1/metabolismo , Neoplasias Hepáticas/metabolismo , Lisina/química , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Proteoma/metabolismo , Transcriptoma
7.
Mol Cell Biol ; 37(4)2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-27895153

RESUMO

Repair of damaged DNA is critical for maintenance of genetic information. In eukaryotes, DNA double-strand breaks (DSBs) are recognized by the Ku70-Ku80 heterodimer, which then recruits proteins that mediate repair by nonhomologous end joining (NHEJ). Prolonged retention of Ku70/80 at DSBs prevents completion of repair, however, with ubiquitylation of Ku80 having been implicated in Ku70/80 dissociation from DNA. Here, we identify RNF126 as a ubiquitin ligase that is recruited to DSBs and ubiquitylates Ku80, with UBE2D3 serving as an E2 enzyme. Knockdown of RNF126 prevented Ku70/80 dissociation from DSBs and inhibited break repair. Attenuation of Ku80 ubiquitylation by replacement of ubiquitylation site lysines with arginine residues delayed Ku70/80 release from chromatin after DSB induction by genotoxic insults. Together, our data indicate that RNF126 is a novel regulator of NHEJ that promotes completion of DNA repair by ubiquitylating Ku80 and releasing Ku70/80 from damaged DNA.


Assuntos
Reparo do DNA por Junção de Extremidades , Autoantígeno Ku/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Células HEK293 , Células HeLa , Humanos , Camundongos , Modelos Biológicos , Células NIH 3T3 , Multimerização Proteica/efeitos da radiação , Proteólise/efeitos da radiação , Radiação Ionizante , Ubiquitinação/efeitos da radiação
8.
PLoS One ; 11(8): e0160555, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27579980

RESUMO

Metabolomics is a promising avenue for biomarker discovery. Although the quality of metabolomic analyses, especially global metabolomics (G-Met) using mass spectrometry (MS), largely depends on the instrumentation, potential bottlenecks still exist at several basic levels in the metabolomics workflow. Therefore, we established a precise protocol initially for the G-Met analyses of human blood plasma to overcome some these difficulties. In our protocol, samples are deproteinized in a 96-well plate using an automated liquid-handling system, and conducted either using a UHPLC-QTOF/MS system equipped with a reverse phase column or a LC-FTMS system equipped with a normal phase column. A normalization protocol of G-Met data was also developed to compensate for intra- and inter-batch differences, and the variations were significantly reduced along with our normalization, especially for the UHPLC-QTOF/MS data with a C18 reverse-phase column for positive ions. Secondly, we examined the changes in metabolomic profiles caused by the storage of EDTA-blood specimens to identify quality markers for the evaluation of the specimens' pre-analytical conditions. Forty quality markers, including lysophospholipids, dipeptides, fatty acids, succinic acid, amino acids, glucose, and uric acid were identified by G-Met for the evaluation of plasma sample quality and established the equation of calculating the quality score. We applied our quality markers to a small-scale study to evaluate the quality of clinical samples. The G-Met protocols and quality markers established here should prove useful for the discovery and development of biomarkers for a wider range of diseases.


Assuntos
Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Metabolômica/métodos , Metabolômica/normas , Biomarcadores/sangue , Cromatografia Líquida/métodos , Cromatografia Líquida/normas , Humanos
9.
J Biol Chem ; 291(39): 20516-29, 2016 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-27496948

RESUMO

In eukaryotic cells, heme production is tightly controlled by heme itself through negative feedback-mediated regulation of nonspecific 5-aminolevulinate synthase (ALAS1), which is a rate-limiting enzyme for heme biosynthesis. However, the mechanism driving the heme-dependent degradation of the ALAS1 protein in mitochondria is largely unknown. In the current study, we provide evidence that the mitochondrial ATP-dependent protease ClpXP, which is a heteromultimer of CLPX and CLPP, is involved in the heme-dependent degradation of ALAS1 in mitochondria. We found that ALAS1 forms a complex with ClpXP in a heme-dependent manner and that siRNA-mediated suppression of either CLPX or CLPP expression induced ALAS1 accumulation in the HepG2 human hepatic cell line. We also found that a specific heme-binding motif on ALAS1, located at the N-terminal end of the mature protein, is required for the heme-dependent formation of this protein complex. Moreover, hemin-mediated oxidative modification of ALAS1 resulted in the recruitment of LONP1, another ATP-dependent protease in the mitochondrial matrix, into the ALAS1 protein complex. Notably, the heme-binding site in the N-terminal region of the mature ALAS1 protein is also necessary for the heme-dependent oxidation of ALAS1. These results suggest that ALAS1 undergoes a conformational change following the association of heme to the heme-binding motif on this protein. This change in the structure of ALAS1 may enhance the formation of complexes between ALAS1 and ATP-dependent proteases in the mitochondria, thereby accelerating the degradation of ALAS1 protein to maintain appropriate intracellular heme levels.


Assuntos
5-Aminolevulinato Sintetase/metabolismo , Heme/metabolismo , Mitocôndrias/enzimologia , Proteólise , 5-Aminolevulinato Sintetase/genética , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Heme/genética , Células Hep G2 , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oxirredução
10.
J Biol Chem ; 291(12): 6316-30, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26786103

RESUMO

B lymphocyte-induced maturation protein 1 (Blimp-1) encoded by Prdm1 is a master regulator of plasma cell differentiation. The transcription factor Bach2 represses Blimp-1 expression in B cells to stall terminal differentiation, by which it supports reactions such as class switch recombination of the antibody genes. We found that histones H3 and H4 around the Prdm1 intron 5 Maf recognition element were acetylated at higher levels in X63/0 plasma cells expressing Blimp-1 than in BAL17 mature B cells lacking its expression. Conversely, methylation of H3-K9 was lower in X63/0 cells than BAL17 cells. Purification of the Bach2 complex in BAL17 cells revealed its interaction with histone deacetylase 3 (HDAC3), nuclear co-repressors NCoR1 and NCoR2, transducin ß-like 1X-linked (Tbl1x), and RAP1-interacting factor homolog (Rif1). Chromatin immunoprecipitation confirmed the binding of HDAC3 and Rif1 to the Prdm1 locus. Reduction of HDAC3 or NCoR1 expression by RNA interference in B cells resulted in an increased Prdm1 mRNA expression. Bach2 is suggested to cooperate with HDAC3-containing co-repressor complexes in B cells to regulate the stage-specific expression of Prdm1 by writing epigenetic modifications at the Prdm1 locus.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Inativação Gênica , Histona Desacetilases/fisiologia , Fatores de Transcrição/genética , Acetilação , Animais , Linfócitos B , Linhagem Celular Tumoral , Epigênese Genética , Células HEK293 , Histonas/metabolismo , Humanos , Camundongos , Correpressor 1 de Receptor Nuclear/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a Telômeros/metabolismo , Fatores de Transcrição/metabolismo
11.
Mol Cell Biol ; 36(3): 407-20, 2016 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-26572828

RESUMO

The KEAP1-NRF2 system plays a central role in cytoprotection. NRF2 is stabilized in response to electrophiles and activates transcription of antioxidant genes. Although robust induction of NRF2 target genes confers resistance to oxidative insults, how NRF2 triggers transcriptional activation after binding to DNA has not been elucidated. To decipher the molecular mechanisms underlying NRF2-dependent transcriptional activation, we purified the NRF2 nuclear protein complex and identified the Mediator subunits as NRF2 cofactors. Among them, MED16 directly associated with NRF2. Disruption of Med16 significantly attenuated the electrophile-induced expression of NRF2 target genes but did not affect hypoxia-induced gene expression, suggesting a specific requirement for MED16 in NRF2-dependent transcription. Importantly, we found that 75% of NRF2-activated genes exhibited blunted inductions by electrophiles in Med16-deficient cells compared to wild-type cells, which strongly argues that MED16 is a major contributor supporting NRF2-dependent transcriptional activation. NRF2-dependent phosphorylation of the RNA polymerase II C-terminal domain was absent in Med16-deficient cells, suggesting that MED16 serves as a conduit to transmit NRF2-activating signals to RNA polymerase II. MED16 indeed turned out to be essential for cytoprotection against oxidative insults. Thus, the KEAP1-NRF2-MED16 axis has emerged as a new regulatory pathway mediating the antioxidant response through the robust activation of NRF2 target genes.


Assuntos
Regulação da Expressão Gênica , Complexo Mediador/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Mapas de Interação de Proteínas , Animais , Linhagem Celular , Proliferação de Células , Técnicas de Inativação de Genes , Humanos , Fígado/metabolismo , Complexo Mediador/química , Complexo Mediador/genética , Camundongos , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Fosforilação , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase II/metabolismo , Transdução de Sinais
12.
Transplantation ; 99(7): 1349-55, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25803499

RESUMO

BACKGROUND: Islet isolation currently requires collagenase, neutral protease and other components. Thermolysin (TL) from Bacillus thermoproteolyticus is the gold standard neutral protease. However, we speculated that neutral protease derived from Clostridium histolyticum (Ch; ChNP) would be biologically superior for islet isolation. Tryptic-like activity has also been reported to be important. Therefore, we focused on clostripain (CP), since it is one of the main proteases in Clostridium histolyticum which possesses tryptic-like activity. We then examined the synergistic effects of highly purified ChNP and CP on rat islet isolation. METHODS: The same amount of collagenase was used in all four groups (TL, ChNP, TL+CP and ChNP+CP; n = 12/group). The efficiency was evaluated by the islet yield and function. An immunohistochemical analysis, in vitro digestion assay for each enzyme component and evaluation of the activation of endogenous exocrine proteases during islet isolation were also performed. RESULTS: The islet yield of the TL group was significantly higher than that of the ChNP group (P < 0.01). The islet yield was dose dependently increased in the ChNP+CP group, but was decreased in the TL + CP group. The islet yield in the ChNP + CP group was significantly higher than that in the TL group, but their islet function was similar. Different specificities for laminin, especially laminin-511, were observed in the TL, ChNP, and CP groups. CONCLUSIONS: Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.


Assuntos
Proteínas de Bactérias/metabolismo , Clostridium histolyticum/enzimologia , Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Ilhotas Pancreáticas/enzimologia , Coleta de Tecidos e Órgãos/métodos , Animais , Proteínas de Bactérias/genética , Clostridium histolyticum/genética , Cisteína Endopeptidases/genética , Endopeptidases/genética , Colagenase Microbiana/isolamento & purificação , Colagenase Microbiana/metabolismo , Ratos , Ratos Endogâmicos Lew , Proteínas Recombinantes/metabolismo , Termolisina/metabolismo , Fatores de Tempo
14.
Biophys J ; 104(7): 1538-45, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23561530

RESUMO

Collagenase H (ColH) from Clostridium histolyticum is a multimodular protein composed of a collagenase module (activator and peptidase domains), two polycystic kidney disease-like domains, and a collagen-binding domain. The interdomain conformation and its changes are very important for understanding the functions of ColH. In this study, small angle x-ray scattering and limited proteolysis were employed to reveal the interdomain arrangement of ColH in solution. The ab initio beads model indicated that ColH adopted a tapered shape with a swollen head. Under calcium-chelated conditions (with EGTA), the overall structure was further elongated. The rigid body model indicated that the closed form of the collagenase module was preferred in solution. The limited proteolysis demonstrated that the protease sensitivity of ColH was significantly increased under the calcium-chelated conditions, and that the digestion mainly occurred in the domain linker regions. Fluorescence measurements with a fluorescent dye were performed with the limited proteolysis products after separation. The results indicated that the limited proteolysis products exhibited fluorescence similar to that of the full-length ColH. These findings suggested that the conformation of full-length ColH in solution is the elongated form, and this form is calcium-dependently maintained at the domain linker regions.


Assuntos
Cálcio/metabolismo , Colagenases/química , Colagenases/metabolismo , Clostridium histolyticum/enzimologia , Modelos Moleculares , Conformação Proteica , Proteólise , Espalhamento a Baixo Ângulo , Soluções , Difração de Raios X
15.
J Biol Chem ; 288(19): 13592-601, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23539621

RESUMO

BACKGROUND: MATII biosynthesizes AdoMet, which supplies methyl group for methylation of molecules, including histone. RESULTS: MATII interacts with histone methyltransferase SETDB1 and inhibits COX-2 gene expression. CONCLUSION: AdoMet synthesis and histone methylation are coupled on chromatin by a physical interaction of MATII and SETDB1 at the MafK target genes. SIGNIFICANCE: MATII may be important for both gene-specific and epigenome-wide regulation of histone methylation. Methionine adenosyltransferase (MAT) synthesizes S-adenosylmethionine (AdoMet), which is utilized as a methyl donor in transmethylation reactions involving histones. MATIIα, a MAT isozyme, serves as a transcriptional corepressor in the oxidative stress response and forms the AdoMet-integrating transcription regulation module, affecting histone methyltransferase activities. However, the identities of genes regulated by MATIIα or its associated methyltransferases are unclear. We show that MATIIα represses the expression of cyclooxygenase 2 (COX-2), encoded by Ptgs2, by specifically interacting with histone H3K9 methyltransferase SETDB1, thereby promoting the trimethylation of H3K9 at the COX-2 locus. We discuss both gene-specific and epigenome-wide functions of MATIIα.


Assuntos
Ciclo-Oxigenase 2/genética , Repressão Enzimática , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Metionina Adenosiltransferase/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Elementos Facilitadores Genéticos , Técnicas de Silenciamento de Genes , Inativação Gênica , Heme Oxigenase-1/genética , Humanos , Proteínas de Membrana/genética , Metionina Adenosiltransferase/genética , Metilação , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/genética , Transcriptoma
16.
Subcell Biochem ; 61: 105-18, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23150248

RESUMO

Histone and DNA methyltransferases utilize S-adenosyl-L-methionine (SAM), a key intermediate of sulfur amino acid metabolism, as a donor of methyl group. SAM is biosynthesized by methionine adenosyltransferase (MAT) using two substrates, methionine and ATP. Three distinct forms of MAT (MATI, MATII and MATIII), encoded by two distinct genes (MAT1A and MAT2A), have been identified in mammals. MATII consists of α2 catalytic subunit encoded by MAT2A and ß regulatory subunit encoded by MAT2B, but the physiological function of the ß subunit is not clear. MafK is a member of Maf oncoproteins and functions as both transcription activator and repressor by forming diverse heterodimers to bind to DNA elements termed Maf recognition elements. Proteomics analysis of MafK-interactome revealed its interaction with both MATIIα and MATIIß. They are recruited specifically to MafK target genes and are required for their repression by MafK and its partner Bach1. Because the catalytic activity of MATIIα is required for the MafK target gene repression, MATIIα is suggested to provide SAM locally on chromatin where it is recruited. One of the unexpected features of MATII is that MATIIα interacts with many chromatin-related proteins of diverse functions such as histone modification, chromatin remodeling, transcription regulation, and nucleo-cytoplasmic transport. MATIIα appears to generate multiple, heterogenous regulatory complexes where it provides SAM. Considering their function, the heterooligomer of MATIIα and ß is named SAMIT (SAM-integrating transcription) module within their interactome where it serves SAM for nuclear methyltransferases.


Assuntos
Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Epigênese Genética , Metionina Adenosiltransferase/metabolismo , S-Adenosilmetionina/biossíntese , Animais , Núcleo Celular/enzimologia , Cromatina/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Epigenômica/métodos , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Fator de Transcrição MafK/metabolismo , Transcrição Gênica
17.
Mol Cell ; 41(5): 554-66, 2011 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-21362551

RESUMO

Protein methylation pathways comprise methionine adenosyltransferase (MAT), which produces S-adenosylmethionine (SAM) and SAM-dependent substrate-specific methyltransferases. However, the function of MAT in the nucleus is largely unknown. MafK represses or activates expression of heme oxygenase-1 (HO-1) gene, depending on its heterodimer partners. Proteomics analysis of MafK revealed its interaction with MATIIα, a MAT isozyme. MATIIα was localized in nuclei and found to form a dense network with chromatin-related proteins including Swi/Snf and NuRD complexes. MATIIα was recruited to Maf recognition element (MARE) at HO-1 gene. When MATIIα was knocked down in murine hepatoma cell line, expression of HO-1 was derepressed at both basal and induced levels. The catalytic activity of MATIIα, as well as its interacting factors such as MATIIß, BAF53a, CHD4, and PARP1, was required for HO-1 repression. MATII serves as a transcriptional corepressor of MafK by interacting with chromatin regulators and supplying SAM for methyltransferases.


Assuntos
Regulação Neoplásica da Expressão Gênica , Metionina Adenosiltransferase/metabolismo , Proteínas Proto-Oncogênicas c-maf/metabolismo , Transcrição Gênica , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Heme Oxigenase-1/biossíntese , Histonas/metabolismo , Humanos , Isoenzimas/química , Neoplasias Hepáticas/metabolismo , Camundongos , Proteínas Oncogênicas/metabolismo , Proteômica/métodos
18.
J Biol Chem ; 285(31): 23581-9, 2010 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-20501657

RESUMO

Bach1 is a member of the basic leucine zipper transcription factor family, and the Bach1/small Maf heterodimer specifically represses transcriptional activity directed by the Maf recognition element (MARE). Because Bach1 is a repressor of the oxidative stress response, we examined the function(s) of Bach1 in keratinocytes subjected to oxidative stress. Oxidative stress induced by H(2)O(2) led to an increase in MARE activity and expression of heme oxygenase-1 (HO-1), an inducible antioxidant defense enzyme. Bach1 depletion by small interfering RNAs or by deletion of Bach1 enhanced HO-1 expression in the absence of H(2)O(2), indicating that Bach1 is a critical repressor of HO-1 in keratinocytes. Although Bach1-deficient or -reduced keratinocytes expressed higher levels of HO-1 than control cells in response to H(2)O(2), Bach1 down-regulation did not attenuate the production of reactive oxygen species by H(2)O(2). In contrast, Bach1 overexpression abolished HO-1 induction by H(2)O(2), which led to increased reactive oxygen species accumulation. HO-1 was induced during keratinocyte differentiation, but MARE activity did not change during differentiation. Furthermore, Bach1 overexpression did not inhibit differentiation-associated induction of HO-1 expression, suggesting that HO-1 induction in differentiation is independent of Bach1. Thus, in response to oxidative stress, Bach1 regulates the oxidation state through the negative control of HO-1 expression prior to terminal keratinocyte differentiation. However, Bach1-mediated repression is negated during keratinocyte differentiation.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação Enzimológica da Expressão Gênica , Heme Oxigenase-1/metabolismo , Queratinócitos/enzimologia , Animais , Animais Recém-Nascidos , Antioxidantes/metabolismo , Diferenciação Celular , Células Cultivadas , Epiderme/metabolismo , Peróxido de Hidrogênio/farmacologia , Queratinócitos/citologia , Camundongos , Camundongos Endogâmicos ICR , Modelos Biológicos , Estresse Oxidativo
19.
Nat Struct Mol Biol ; 15(12): 1246-54, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19011633

RESUMO

Cellular senescence is one of the key strategies to suppress expansion of cells with mutations. Senescence is induced in response to genotoxic and oxidative stress. Here we show that the transcription factor Bach1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1), which inhibits oxidative stress-inducible genes, is a crucial negative regulator of oxidative stress-induced cellular senescence. Bach1-deficient murine embryonic fibroblasts showed a propensity to undergo more rapid and profound p53-dependent premature senescence than control wild-type cells in response to oxidative stress. Bach1 formed a complex that contained p53, histone deacetylase 1 and nuclear co-repressor N-coR. Bach1 was recruited to a subset of p53 target genes and contributed to impeding p53 action by promoting histone deacetylation. Because Bach1 is regulated by oxidative stress and heme, our data show that Bach1 connects oxygen metabolism and cellular senescence as a negative regulator of p53.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Senescência Celular , Cromatina/metabolismo , Estresse Oxidativo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Contagem de Células , Proliferação de Células , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Histona Desacetilase 1 , Histona Desacetilases/metabolismo , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Correpressor 1 de Receptor Nuclear , Ligação Proteica , Proteínas Repressoras/metabolismo
20.
J Biol Chem ; 282(47): 34365-71, 2007 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-17901053

RESUMO

Ferritin gene transcription is regulated by heme as is ferritin mRNA translation, which is mediated by the well studied mRNA.IRE/IRP protein complex. The heme-sensitive DNA sequence in ferritin genes is the maf recognition/antioxidant response element present in several other genes that are induced by heme and repressed by Bach1. We now report that chromatin immunoprecipitated with Bach1 antiserum contains ferritin DNA sequences. In addition, overexpression of Bach1 protein in the transfected cells decreased ferritin expression, indicating insufficient endogenous Bach1 for full repression; decreasing Bach1 with antisense RNA increased ferritin expression. Thioredoxin reductase1, a gene that also contains a maf recognition/antioxidant response element but is less studied, responded similarly to ferritin, as did the positive controls heme oxygenase1 and NADP(H) quinone (oxido) reductase1. Bach1-DNA promoter interactions in cells were confirmed in vitro with soluble, recombinant Bach1 protein and revealed a quantitative range of Bach1/DNA stabilities: ferritin L approximately ferritin H approximately beta-globin, beta-globin approximately 2-fold >heme oxygenase1 = quinone reductase beta-globin approximately 4-fold >thioredoxin reductase1. Such results indicate the possibility that modulation of cellular Bach1 concentrations will have variable effects among the genes coordinately regulated by maf recognition/antioxidant response elements in iron/oxygen/antioxidant metabolism.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Ferritinas/biossíntese , Heme Oxigenase-1/biossíntese , Heme/metabolismo , NAD(P)H Desidrogenase (Quinona)/biossíntese , Proteínas Repressoras/metabolismo , Tiorredoxina Redutase 1/biossíntese , Animais , Antioxidantes/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linhagem Celular Tumoral , Ferritinas/genética , Heme/farmacologia , Heme Oxigenase-1/genética , Ferro/metabolismo , Proteínas Reguladoras de Ferro/genética , Proteínas Reguladoras de Ferro/metabolismo , Camundongos , NAD(P)H Desidrogenase (Quinona)/genética , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , RNA Antissenso/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Elementos de Resposta/fisiologia , Tiorredoxina Redutase 1/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia
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