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1.
Front Neurosci ; 14: 562292, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33192249

RESUMO

A recurrent de novo mutation in the transcriptional corepressor CTBP1 is associated with neurodevelopmental disabilities in children (Beck et al., 2016, 2019; Sommerville et al., 2017). All reported patients harbor a single recurrent de novo heterozygous missense mutation (p.R342W) within the cofactor recruitment domain of CtBP1. To investigate the transcriptional activity of the pathogenic CTBP1 mutant allele in physiologically relevant human cell models, we generated induced pluripotent stem cells (iPSC) from the dermal fibroblasts derived from patients and normal donors. The transcriptional profiles of the iPSC-derived "early" neurons were determined by RNA-sequencing. Comparison of the RNA-seq data of the neurons from patients and normal donors revealed down regulation of gene networks involved in neurodevelopment, synaptic adhesion and anti-viral (interferon) response. Consistent with the altered gene expression patterns, the patient-derived neurons exhibited morphological and electrophysiological abnormalities, and susceptibility to viral infection. Taken together, our studies using iPSC-derived neuron models provide novel insights into the pathological activities of the CTBP1 p.R342W allele.

2.
Neurogenetics ; 20(3): 129-143, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31041561

RESUMO

We previously reported a pathogenic de novo p.R342W mutation in the transcriptional corepressor CTBP1 in four independent patients with neurodevelopmental disabilities [1]. Here, we report the clinical phenotypes of seven additional individuals with the same recurrent de novo CTBP1 mutation. Within this cohort, we identified consistent CtBP1-related phenotypes of intellectual disability, ataxia, hypotonia, and tooth enamel defects present in most patients. The R342W mutation in CtBP1 is located within a region implicated in a high affinity-binding cleft for CtBP-interacting proteins. Unbiased proteomic analysis demonstrated reduced interaction of several chromatin-modifying factors with the CtBP1 W342 mutant. Genome-wide transcriptome analysis in human glioblastoma cell lines expressing -CtBP1 R342 (wt) or W342 mutation revealed changes in the expression profiles of genes controlling multiple cellular processes. Patient-derived dermal fibroblasts were found to be more sensitive to apoptosis during acute glucose deprivation compared to controls. Glucose deprivation strongly activated the BH3-only pro-apoptotic gene NOXA, suggesting a link between enhanced cell death and NOXA expression in patient fibroblasts. Our results suggest that context-dependent relief of transcriptional repression of the CtBP1 mutant W342 allele may contribute to deregulation of apoptosis in target tissues of patients leading to neurodevelopmental phenotypes.


Assuntos
Oxirredutases do Álcool/genética , Proteínas de Ligação a DNA/genética , Mutação de Sentido Incorreto , Adolescente , Oxirredutases do Álcool/metabolismo , Alelos , Apoptose , Ataxia/complicações , Ataxia/genética , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Criança , Pré-Escolar , Cromatina/química , Proteínas de Ligação a DNA/metabolismo , Feminino , Fibroblastos/metabolismo , Glioblastoma/genética , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/genética , Masculino , Hipotonia Muscular/complicações , Hipotonia Muscular/genética , Fenótipo , Ligação Proteica , Proteômica , Anormalidades Dentárias/complicações , Anormalidades Dentárias/genética , Adulto Jovem
3.
J Virol ; 90(2): 1070-9, 2016 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-26559831

RESUMO

UNLABELLED: The cell-transforming activity of human adenovirus 5 (hAd5) E1A is mediated by the N-terminal half of E1A, which interacts with three different major cellular protein complexes, p300/CBP, TRRAP/p400, and pRb family members. Among these protein interactions, the interaction of pRb family proteins with conserved region 2 (CR2) of E1A is known to promote cell proliferation by deregulating the activities of E2F family transcription factors. The functional consequences of interaction with the other two protein complexes in regulating the transforming activity of E1A are not well defined. Here, we report that the E1A N-terminal region also interacted with the cellular proto-oncoprotein c-MYC and the homolog of enhancer of yellow 2 (ENY2). Our results suggested that these proteins interacted with an essential E1A transforming domain spanning amino acid residues 26 to 35 which also interacted with TRRAP and p400. Small interfering RNA (siRNA)-mediated depletion of TRRAP reduced c-MYC interaction with E1A, while p400 depletion did not. In contrast, depletion of TRRAP enhanced ENY2 interaction with E1A, suggesting that ENY2 and TRRAP may interact with E1A in a competitive manner. The same E1A region additionally interacted with the constituents of a deubiquitinase complex consisting of USP22, ATXN7, and ATXN7L3 via TRRAP. Acute short hairpin RNA (shRNA)-mediated depletion of c-MYC reduced the E1A transforming activity, while depletion of ENY2 and MAX did not. These results suggested that the association of c-MYC with E1A may, at least partially, play a role in the E1A transformation activity, independently of MAX. IMPORTANCE: The transforming region of adenovirus E1A consists of three short modules which complex with different cellular protein complexes. The mechanism by which one of the transforming modules, CR2, promotes cell proliferation, through inactivating the activities of the pRb family proteins, is better understood than the activities of the other domains. Our analysis of the E1A proteome revealed the presence of the proto-oncoprotein c-MYC and of ENY2. We mapped these interactions to a critical transforming module of E1A that was previously known to interact with the scaffolding molecule TRRAP and the E1A-binding protein p400. We showed that c-MYC interacted with E1A through TRRAP, while ENY2 interacted with it independently. The data reported here indicated that depletion of c-MYC in normal human cells reduced the transforming activity of E1A. Our result raises a novel paradigm in oncogenic transformation by a DNA viral oncogene, the E1A gene, that may exploit the activity of a cellular oncogene, the c-MYC gene, in addition to inactivation of the tumor suppressors, such as pRb.


Assuntos
Proteínas E1A de Adenovirus/metabolismo , Adenovírus Humanos/fisiologia , Interações Hospedeiro-Patógeno , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linhagem Celular , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas , Fatores de Transcrição/metabolismo
4.
Virology ; 483: 21-31, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25965792

RESUMO

Adenovirus-mediated apoptosis was suppressed when cellular anti-apoptosis proteins (BCL-2 and BCL-xL) were substituted for the viral E1B-19K. For unbiased proteomic analysis of proteins targeted by BCL-xL in adenovirus-infected cells and to visualize the interactions with target proteins, BCL-xL was targeted to cytosolic inclusion bodies utilizing the orthoreovirus µNS protein sequences. The chimeric protein was localized in non-canonical cytosolic factory-like sites and promoted survival of virus-infected cells. The BCL-xL-associated proteins were isolated from the cytosolic inclusion bodies in adenovirus-infected cells and analyzed by LC-MS. These proteins included BAX, BAK, BID, BIK and BIM as well as mitochondrial proteins such as prohibitin 2, ATP synthase and DNA-PKcs. Our studies suggested that in addition to the interaction with various pro-apoptotic proteins, the association with certain mitochondrial proteins such as DNA-PKcs and prohibitins might augment the survival function of BCL-xL in virus infected cells.


Assuntos
Adenoviridae/crescimento & desenvolvimento , Corpos de Inclusão/virologia , Proteínas Mitocondriais/metabolismo , Mapeamento de Interação de Proteínas , Proteína bcl-X/metabolismo , Apoptose , Linhagem Celular , Sobrevivência Celular , Cromatografia Líquida , Humanos , Espectrometria de Massas , Ligação Proteica
5.
Genes Cancer ; 5(1-2): 31-40, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24955216

RESUMO

C-terminal binding protein (CtBP) family transcriptional corepressors include CtBP1 and CtBP2. While CtBP1 and CtBP2 share significant amino acid sequence homology, CtBP2 possesses a unique N-terminal domain that is modified by acetylation and contributes to exclusive nuclear localization. Although CtBP1 and CtBP2 are functionally redundant for certain activities during vertebrate development, they also perform unique functions. Previous studies have identified several CtBP1-interacting proteins that included other transcriptional corepressors, DNA-binding repressors and histone modifying enzymatic components such as the histone deacetylases and the histone demethylase LSD-1. Here, we carried out an unbiased proteomic analysis of CtBP2-associated proteins and discovered the association of several components of the CtBP1 proteome as well as novel interactions. The CtBP2 proteome contained components of the NuRD complex and the E2F family member E2F7. E2F7 interacted with the hydrophobic cleft region of CtBP1 and CtBP2 through a prototypical CtBP binding motif, PIDLS. E2F7 repressed E2F1 transcription, inhibited cell proliferation in a CtBP-dependent fashion. Our study identified CtBP as a corepressor of E2F7 and as a regulator of DNA damage response.

6.
Cancer Gene Ther ; 21(6): 228-237, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24874842

RESUMO

Human adenovirus (HAdV) vectors are intensely investigated for virotherapy of a wide variety of human cancers. Here, we have evaluated the effect of two apoptogenic HAdV5 vectors in an immunocompetent Syrian hamster animal model of head and neck cancer. We established two cell lines of hamster cheek pouch squamous cell carcinomas, induced by treatment with 9,10-dimethyl-1,2-benzanthracene. These cell lines, when infected with HAdV5 mutants lp11w and lp11w/Δ55 K (which are defective in the expression of either E1B-19 K alone or both E1B-19 K and E1B-55 K proteins) exhibited enhanced apoptotic and cytotoxic responses. The cheek pouch tumor cells transplanted either subcutaneously at the flanks or in the cheek pouches of hamsters readily formed tumors. Intratumoral administration of HAdV5-E1B mutants efficiently suppressed the growth of tumors at both sites. Histological examination of orthotopic tumors revealed reduced vascularity and the expression of the viral fiber antigen in virus-administered cheek pouch tumors. These tumors also exhibited increased caspase-3 levels, suggesting that virus-induced apoptosis may contribute to tumor growth suppression. Our results suggest that the apoptogenic HAdV5 vectors may have utility for the treatment of human head and neck cancers.


Assuntos
Adenovírus Humanos/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Terapia Viral Oncolítica/métodos , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Proteínas E1B de Adenovirus/genética , Animais , Apoptose/genética , Carcinoma de Células Escamosas/induzido quimicamente , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Neoplasias de Cabeça e Pescoço/induzido quimicamente , Masculino , Mesocricetus , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Virol ; 87(13): 7781-6, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23637414

RESUMO

The adenovirus E1A C-terminal region restrains oncogenic transformation through interaction with three distinct cellular protein complexes that include the DYRK1A/1B/HAN11 complex. The E6 proteins of beta-human papillomaviruses (beta-HPVs) also interact with the DYRK1/HAN11 complex. A variant of HPV5 E6 frequently found in epidermodysplasia verruciformis skin lesions interacted less efficiently with DYRK1A/HAN11. The E6 variant and E7 of HPV5 efficiently coimmortalized primary epithelial cells, suggesting that naturally arising variants may contribute potential oncogenic activities of beta-HPV E6 proteins.


Assuntos
Proteínas E1A de Adenovirus/metabolismo , Betapapillomavirus/metabolismo , Transformação Celular Neoplásica/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas E1A de Adenovirus/genética , Sequência de Aminoácidos , Western Blotting , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Proteínas Oncogênicas Virais/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Homologia de Sequência , Replicação Viral/genética , Quinases Dyrk
8.
J Virol ; 87(6): 3425-34, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23302885

RESUMO

The adenovirus large E1A (L-E1A) protein is a prototypical transcriptional activator, and it functions through the action of a conserved transcriptional activation domain, CR3. CR3 interacts with a mediator subunit, MED23, that has been linked to the transcriptional activity of CR3. Our unbiased proteomic analysis revealed that human adenovirus 5 (HAdv5) L-E1A was associated with many mediator subunits. In MED23-depleted cells and in Med23 knockout (KO) cells, L-E1A was deficient in association with other mediator subunits, suggesting that MED23 links CR3 with the mediator complex. Short interfering RNA (siRNA)-mediated depletion of several mediator subunits suggested differential effects of various subunits on transcriptional activation of HAdv5 early genes. In addition to MED23, mediator subunits such as MED14 and MED26 were also essential for the transcription of HAdv5 early genes. The L-E1A proteome contained MED26-associated super elongation complex. The catalytic component of the elongation complex, CDK9, was important for the transcriptional activity of L-E1A and HAdv5 replication. Our results suggest that L-E1A-mediated transcriptional activation involves a transcriptional elongation step, like HIV Tat, and constitutes a therapeutic target for inhibition of HAdv replication.


Assuntos
Proteínas E1A de Adenovirus/metabolismo , Adenovírus Humanos/genética , Regulação Viral da Expressão Gênica , Complexo Mediador/metabolismo , Transcrição Gênica , Humanos
9.
J Virol ; 84(6): 2719-31, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20053746

RESUMO

The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.


Assuntos
Proteínas E1A de Adenovirus/metabolismo , Betapapillomavirus/fisiologia , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas E1A de Adenovirus/genética , Sequência de Aminoácidos , Animais , Betapapillomavirus/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Oncogênicas Virais/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologia , Quinases Dyrk
10.
Genes Cancer ; 1(9): 964-71, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21779475

RESUMO

BCL-2/E1B-19 kDa-interacting protein 3 (BNIP3) is a BH3-only mitochondrial protein. Expression of BNIP3 is strongly stimulated by hypoxia. Up-regulation of BNIP3 has been detected in several human carcinomas including carcinomas of the lung and breast. The significance of BNIP3 overexpression in these cancers is not known. To determine whether BNIP3 plays a role in tumor growth, we generated A549 lung carcinoma cells that overexpressed BNIP3 and examined their ability to form tumors in the mouse xenograft model. All cell lines that overexpressed BNIP3 formed larger tumors compared to the parental or vector-transformed A549 cells. Breast carcinoma cell lines that overexpressed BNIP3 also induced tumors in athymic mice in the absence of hormone administration, while the parental cell line did not. Stable shRNA-mediated knockdown of endogenous BNIP3 severely impaired the tumorigenic activity of A549 cells. The tumor growth-enhancing activity was reduced by deletion of the BH3 domain of BNIP3. Expression of a dominant-negative mutant of BNIP3 lacking the C-terminal transmembrane domain also inhibited the tumorigenic potential of A549 cells. These results suggest that BNIP3 plays a fundamental role in the development of certain solid tumors such as the lung and breast carcinomas.

11.
BMC Mol Biol ; 10: 89, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19754958

RESUMO

BACKGROUND: Proteins of the C-terminal binding protein (CtBP) family, CtBP1 and CtBP2 are closely related transcriptional regulators that are coded by two different gene loci in the vertebrate genomes. They perform redundant and unique functions during animal development. CtBP proteins mediate their transcriptional function through interaction with various DNA-binding repressors that contain PLDLS-like motifs and chromatin modifying enzymes, such as class I histone deacetylases (HDAC) that do not contain such motifs. The N-terminal region of CtBP1/2 forms a hydrophobic cleft and is involved in interaction with both PLDLS-containing factors and non-PLDLS factors. CtBP proteins function as dimers to mediate transcriptional repression and dimerization is modulated by specific binding to NAD/NADH. RESULTS: In this study, we have investigated the role of dimerization of CtBP2 in recruitment of PLDLS-motif cofactors and non-PLDLS cofactors. Our results indicate that mutations in CtBP2 that interfere with dimerization abolish CtBP2 interaction with most cellular factors, except the PLDLS-motif factor zinc-finger E-box binding homeobox (ZEB) and the non-PLDLS factor HDAC2. Unlike most PLDLS-containing CtBP-binding proteins, ZEB contains three PLDLS-like motifs and all three contribute to the interaction with the CtBP2 monomer. Despite the ability to interact with ZEB and HDAC, the CtBP2 monomer fails to mediate ZEB-dependent transcriptional repression. The lack of repression activity of the CtBP2 monomer is correlated with the competition between ZEB and HDAC for interaction with the CtBP2 monomer. CONCLUSION: These results suggest a competition between the canonical PLDLS-motif factors such as E1A and non-PLDLS factor HDAC for interaction with CtBP. They also indicate that the affinity for the CtBP monomer may be determined by the number as well as amino acid sequence compositions of the PLDLS-like motifs. Our results are consistent with a model that the CtBP2 dimer may interact with a PLDLS-containing repressor through one monomer and recruit HDAC and other chromatin modifying enzymes through the second monomer in the CtBP2 dimer.


Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Histona Desacetilases/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Oxirredutases do Álcool/genética , Motivos de Aminoácidos , Linhagem Celular , Proteínas Correpressoras , Dimerização , Histona Desacetilase 2 , Histona Desacetilases/genética , Proteínas de Homeodomínio/genética , Humanos , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco
12.
Virology ; 392(1): 62-72, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19631957

RESUMO

Head and neck squamous cell carcinomas (HNSCC) are one of the leading causes of cancer deaths world wide. Up-regulation of the epidermal growth factor receptor (EGFR) and BCL-2 family anti-apoptosis proteins in these cancers is linked to aggressive tumor growth, metastasis and chemoresistance. Infection of two HNSCC cell lines, SCC25 and CAL27 by an Ad5 mutant (lp11w) defective in coding for the viral anti-apoptosis protein, E1B-19K efficiently induced apoptotic cell death. In cells infected with lp11w there was a dramatic down-regulation of EGFR by apoptosis-dependent and -independent mechanisms. The levels of the anti-apoptotic proteins BCL-2, BCL-xL and MCL-1 were also down-regulated in lp11w-infected cells compared to uninfected or Ad5-RM infected cells. Infection with lp11w also enhanced sensitivity of the HNSCC cells to the chemotherapeutic drug cisplatin. Our results suggest that adenoviral vectors defective in E1B-19K would be valuable for efficient down-regulation of cell survival proteins and EGFR in epithelial cancers and could be exploited as oncolytic agents to treat HNSCCs.


Assuntos
Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Apoptose , Sequência de Bases , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Linhagem Celular Tumoral , Sobrevivência Celular , DNA Viral/genética , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Vetores Genéticos , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides , Terapia Viral Oncolítica , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
13.
Oncogene ; 27 Suppl 1: S114-27, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19641497

RESUMO

The BNIP3 subfamily of BH3-only proteins consists of BNIP3 and BNIP3-like (BNIP3L) proteins. These proteins form stable homodimerization complexes that localize to the outer membrane of the mitochondria after cellular stress. This promotes either apoptotic or non-apoptotic cell death such as autophagic cell death. Although the mammalian cells contain both members of this subfamily, the genome of Caenorhabditis elegans codes for a single BNIP3 ortholog, ceBNIP3, which shares homology in the transmembrane (TM) domain and in a conserved region close to the BH3 domain of mammalian BNIP3 protein. The cell death activities of BNIP3 and BNIP3L are determined by either the BH3 domain or the C-terminal TM domain. The TM domain of BNIP3 is unique, as it is capable of autonomous stable dimerization and contributes to mitochondrial localization of BNIP3. In knockout mouse models, BNIP3L was shown to be essential for normal erythrocyte differentiation and hematopoietic homeostasis, whereas BNIP3 plays a role in cellular responses to ischemia/reperfusion injury in the heart. Both BNIP3 and BNIP3L play a role in cellular responses to stress. Under hypoxia, both BNIP3 and BNIP3L expression levels are elevated and contribute to hypoxia-induced cell death. In addition, these proteins play critical roles in disease states. In heart disease, both BNIP3 and BNIP3L play a critical role in cardiomyocyte cell death following ischemic and non-ischemic injuries. In cancer, expression of BNIP3 and BNIP3L is downregulated by promoter hypermethylation or by homozygous deletion of the gene locus in certain cancers, whereas their expression was increased in other cancers. In addition, BNIP3 expression has been correlated with poor prognosis in some cancers. The results reviewed here suggest that BNIP3 and BNIP3L may be novel therapeutic targets for intervention because of their pathological roles in regulating cell death in disease states.


Assuntos
Hipóxia Celular/fisiologia , Proteínas de Membrana/fisiologia , Mitocôndrias , Proteínas Proto-Oncogênicas/fisiologia , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/fisiologia , Artrite Reumatoide/metabolismo , Autofagia/fisiologia , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Morte Celular/fisiologia , Hipóxia Celular/genética , Sequência Conservada , Eritropoese/genética , Eritropoese/fisiologia , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/fisiologia , Proteínas Mitocondriais/fisiologia , Dados de Sequência Molecular , Miócitos Cardíacos/patologia , Neoplasias/etiologia , Neoplasias/genética , Neoplasias/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia
14.
Oncogene ; 27 Suppl 1: S20-9, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19641504

RESUMO

BIK is the founding member of the BH3-only family pro-apoptotic proteins. BIK is predominantly localized in the ER and induces apoptosis through the mitochondrial pathway by mobilizing calcium from the ER to the mitochondria and remodeling the mitochondrial cristae. BIK-mediated apoptosis is mediated by selective activation of BAX. BIK also induces non-apoptotic cell death in certain cell types by unknown mechanisms. BIK is non-essential for animal development, but appears to be functionally redundant for certain developmental functions with BIM. BIK is implicated in the selection of mature B cells in humans. BIK is a pro-apoptotic tumor suppressor in several human tissues and its expression in cancers is prevented by chromosomal deletions encompassing the Bik locus or by epigenetic silencing. BIK appears to be a critical effector in apoptosis induced by toxins, cytokines and virus infection. Several anti-cancer drugs transcriptionally activate Bik gene expression through transcriptional pathways dependent on factors such as E2F and p53 or by removal of epigenetic marks on the chromatin. BIK appears to be a prominent target for anti-cancer drugs that inhibit proteasomal functions. BIK has also been used as a therapeutic molecule in gene therapy-based approaches to treat difficult cancers.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Regulação da Expressão Gênica , Terapia Genética , Humanos , Mamíferos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/fisiologia , Dados de Sequência Molecular , Proteínas de Neoplasias/fisiologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia , Proteína X Associada a bcl-2/metabolismo
15.
Mol Cell Biol ; 28(1): 269-81, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17967884

RESUMO

C-terminal binding protein (CtBP) family proteins CtBP1 and CtBP2 are highly homologous transcriptional corepressors and are recruited by a large number of transcription factors to mediate sequence-specific transcriptional repression. In addition to DNA-binding repressors, the nuclear protein complex of CtBP1 consists of enzymatic constituents such as histone deacetylases (HDAC1/2), histone methyl transferases (HMTases; G9a and GLP), and the lysine-specific demethylase (LSD1). Additionally, CtBPs also recruit the components of the sumoylation machinery. The CtBPs contain two different unique structural elements, a hydrophobic cleft, with which factors that contain motifs related to the E1A PLDLS motif bind, and a surface groove that binds with factors containing motifs related to the sequence RRTGXPPXL (RRT motif). By structure-based functional dissection of CtBP1, we show that the PLDLS-binding cleft region functions as the primary recruitment center for DNA-binding factors and for the core and auxiliary enzymatic constituents of the CtBP1 corepressor complex. We identify HDAC1/2, CoREST/LSD1, and Ubc9 (E2) as the core constituents of the CtBP1 complex, and these components interact with the PLDLS cleft region through non-PLDLS interactions. Among the CtBP core constituents, HDACs contribute predominantly to the repression activity of CtBP1. The auxiliary components include an HMTase complex (G9a/Wiz/CDYL) and two SUMO E3 ligases, HPC2 and PIAS1. The interaction of auxiliary components with CtBP1 is excluded by PLDLS (E1A)-mediated interactions. Although monomeric CtBP1 is proficient in the recruiting of both core and auxiliary components, NAD(H)-dependent dimerization is required for transcriptional repression. We also provide evidence that CtBP1 functions as a platform for sumoylation of cofactors.


Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Oxirredutases do Álcool/deficiência , Oxirredutases do Álcool/genética , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Knockout , Mutação/genética , Ligação Proteica , Proteína SUMO-1/metabolismo , Transativadores/metabolismo , Transcrição Gênica/genética , Fatores de Transcrição de p300-CBP/genética
16.
Oncogene ; 27(10): 1366-75, 2008 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-17873911

RESUMO

The BH3-only protein BIK normally induces apoptotic cell death. Here, we have investigated the role of BCL-2 in BIK-induced cell death using Bcl-2+/+ and Bcl-2-/- mouse embryo fibroblasts. Ectopic expression of BIK in Bcl-2-/- cells resulted in enhanced cell death compared to Bcl-2+/+ cells. In these cells, while caspase-8 was activated, there was no significant activation of caspase-9 and 3. There was no detectable mitochondrial to cytosolic release of cytochrome-c. However, there was significant redistribution of AIF from mitochondria to the nucleus. The extent of BIK-induced cell death was augmented by treatment with the pancaspase inhibitor, zVAD-fmk. The Bcl-2 null cells expressing BIK exhibited autophagic features such as cytosolic vacuoles, punctate distribution of LC3 and enhanced expression of Beclin-1. The survival of BIK-expressing Bcl-2-/- cells was enhanced in the presence of PI3 kinase inhibitors 3-methyladenine and Wortmannin and also by depletion of Atg5 and Beclin-1. Death of BIK-expressing Bcl-2-/- cells treated with zVAD-fmk was increased under caspase-8 depletion. Our results suggest enhanced expression of BIK in the Bcl-2 deficient cells leads to cell death with autophagic features and the extent of such cell death could be increased by inhibition of caspases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Apoptose , Caspases/fisiologia , Genes bcl-2 , Proteínas Mitocondriais/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose , Autofagia/genética , Linhagem Celular Transformada , Células Cultivadas , Camundongos , Estrutura Terciária de Proteína/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia
17.
J Virol ; 81(19): 10486-95, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17652400

RESUMO

Mammalian cells infected with human adenoviruses (Ads) undergo an apoptotic response as a result of expression of the viral E1A proteins, and this process is suppressed by the viral E1B-19K protein. The intermediary steps in the Ad-induced apoptosis pathway are not fully resolved. The apical step in the canonical mammalian apoptosis pathway involves functional activation of one or more of the BH3-only BCL-2 family proapoptotic proteins. Previous reports have suggested that Ad-induced apoptosis may be initiated at checkpoints downstream of the BH3-only proteins. Here, we undertook genetic and biochemical studies to determine the roles of BH3-only proteins in Ad-induced apoptosis. We examined the activities of the cellular antiapoptosis protein BCL-xL and its mutants expressed from the E1B region of the Ad5 genome. Our results showed efficient suppression of Ad-induced apoptosis by a BCL-xL mutant (mt1) deficient in interaction with multidomain proapoptotic proteins BAX and BAK but proficient in interaction with BH3-only proteins, suggesting a role for BH3-only proteins in the initiation of Ad-induced apoptosis. Further, the antiapoptotic activity of BCL-xL mt1 in Ad-infected cells was observed in spite of BAK activation as a consequence of MCL-1 degradation. Analysis of the mRNA levels of various BH3-only members by reverse transcription-PCR revealed prominent activation of the Bik gene. Further, the BIK protein was also modified into an apoptotically enhanced phosphorylated form during the viral infection. In addition to BIK, enhanced level of BIM was observed in Ad-infected cells. Between the two major E1A proteins coded by the 12S and 13S mRNAs, the 13S product appeared to contribute to the activation of these BH3-only members and apoptosis during viral infection. Depletion of BIK by the use of small interfering RNA reduced the level of Ad-induced apoptosis. Our results are consistent with a model that activation of the BH3-only members may initiate Ad-induced apoptosis.


Assuntos
Adenoviridae/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais , Mutação , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Interferente Pequeno/farmacologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
18.
Oncogene ; 26(54): 7544-51, 2007 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-17546044

RESUMO

C-terminal binding proteins (CtBPs) are cellular corepressors that are targeted by adenovirus E1A. A conserved motif of E1A (PLDLS) interacts with an N-terminal hydrophobic cleft of CtBPs. Many cellular cofactors also interact with CtBPs through PLDLS-like motifs. E1A interaction with CtBP2 changed the composition of the CtBP2 protein complex and enhanced CtBP2 acetylation. We have identified a mutant of CtBP2 (M48A) that fails to interact with cellular cofactors while interacting normally with E1A. Other cleft mutations in CtBP2 affected interaction of both cellular cofactors and E1A. The M48A mutant did not repress the cellular E-cadherin promoter but inhibited transactivation mediated by the E1A N-terminal region through interaction with the E1A PLDLS motif. In vitro, E1A enhanced CtBP2 acetylation by p300 via a mechanism involving dissociation of acetylated CtBP2 from p300. E1A enhanced nuclear localization of CtBP1 as well as a cytoplasmically localized acetylation-deficient mutant of CtBP2 (3KR-CtBP2) through PLDLS-dependent interaction. Chromatin immunoprecipitation assays revealed presence of CtBP2 on E-cadherin and c-fos promoters. While E1A did not significantly alter targeting of CtBP2 to the E-cadherin and c-fos promoters, it dramatically enhanced promoter targeting of 3KR-CtBP2. Our results raise a possibility that E1A may gain access to cellular promoters through PLDLS-dependent interaction with CtBPs.


Assuntos
Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/metabolismo , Proteínas do Olho/metabolismo , Acetilação , Proteínas E1A de Adenovirus/genética , Oxirredutases do Álcool , Substituição de Aminoácidos , Animais , Proteínas Correpressoras , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Camundongos , Mutação , Proteínas do Tecido Nervoso , Fosfoproteínas/metabolismo , Transcrição Gênica
19.
J Virol ; 80(4): 2000-12, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16439556

RESUMO

The mechanisms that control cell-to-cell spread of human adenoviruses (Ad) are not well understood. Two early viral proteins, E1B-19K and E3-ADP, appear to have opposing effects since viral mutants that are individually deficient in E1B-19K produce large plaques (G. Chinnadurai, Cell 33:759-766, 1983), while mutants deficient in E3-ADP produce small plaques (A. E. Tollefson et al., J. Virol. 70:2296-2306, 1996) on infected cell monolayers. We have used a genetic strategy to identify different viral genes that influence adenovirus type 5 (Ad5) spread in an epithelial cancer cell line. An Ad5 mutant (dl327; lacking most of the E3 region) with the restricted-spread (small-plaque) phenotype was randomly mutagenized with UV, and 27 large-plaque (lp) mutants were isolated. A combination of analyses of viral proteins and genomic DNA sequences have indicated that 23 mutants contained lesions in the E1B region affecting either 19K or both 19K and 55K proteins. Four other lp mutants contained lesions in early regions E1A and E4, in the early L1 region that codes for the i-leader protein, and in late regions that code for the viral structural proteins, penton base, and fiber. Our results suggest that the requirement of E3-ADP for Ad spread could be readily compensated for by abrogation of the functions of E1B-19K and provide genetic evidence that these two viral proteins influence viral spread in opposing manners. In addition to E1B and E3 proteins, other early and late proteins that regulate viral replication and infectivity also influence lateral viral spread. Our studies have identified novel mutations that could be exploited in designing efficient oncolytic Ad vectors.


Assuntos
Adenovírus Humanos/genética , Adenovírus Humanos/patogenicidade , Genes Virais , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/fisiologia , Proteínas E3 de Adenovirus/genética , Proteínas E3 de Adenovirus/fisiologia , Adenovírus Humanos/fisiologia , Substituição de Aminoácidos , Linhagem Celular , DNA Viral/química , DNA Viral/genética , Humanos , Mutagênese , Mutação Puntual , Raios Ultravioleta , Ensaio de Placa Viral , Proteínas Virais/análise
20.
Diabet Med ; 13(8): 715-9, 1996 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8862945

RESUMO

A total of 105 subjects with impaired glucose tolerance were classified into two groups, 51 subjects with plasma glucose > 11.1 mmol l-1 in one of the blood samplings during OGTT, but at 2 h being less than < 11.1 mmol l-1 were classified as early hyperglycaemics. Fifty-four cases were classified as true IGT, with fasting plasma glucose < 7.8 mmol l-1 and post plasma glucose level between 7.8 and 11.1 mmol l-1. Age and sex matched groups of normals (healthy adults) and NIDDM cases without symptomatic secondary complications were also included in the study. Lipid peroxidation (LPO) product in plasma, erythrocyte, and erythrocyte cell membrane were found to be significantly elevated (p < 0.001) in IGT, early hyperglycaemia and diabetes mellitus while glycosylated haemoglobin was also higher. Antioxidant enzymes superoxide dismutase and catalase were significantly lower in red blood cells obtained from IGT and early hyperglycaemic groups. They were closer to the levels showed in NIDDM confirming that antioxidant deficiency is already present in subjects classified as impaired glucose tolerant. Among the antioxidant scavengers, reduced glutathione (GSH) and ascorbic acid are reduced by 15% and 20% in IGT and NIDDM, respectively. We conclude that antioxidant status is poor in both IGT and NIDDM, suggesting an overlap of frank diabetic state in those classified as IGT. It is possible that antioxidant therapy might retard progression from IGT to NIDDM.


Assuntos
Antioxidantes/análise , Diabetes Mellitus Tipo 2/sangue , Intolerância à Glucose/sangue , Adulto , Glicemia/análise , Estudos de Coortes , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Feminino , Glutationa/sangue , Humanos , Peroxidação de Lipídeos , Masculino , Malondialdeído/sangue , Pessoa de Meia-Idade , Peroxidases/sangue , Valores de Referência , Superóxido Dismutase/sangue , Vitaminas/sangue
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