RESUMO
The ubiquitous MRG/MORF family of proteins is involved in cell senescence, or the terminal loss of proliferative potential, a model for aging and tumor suppression at the cellular level. These proteins are defined by the approximately 20 kDa MRG domain that binds a plethora of transcriptional regulators and chromatin-remodeling factors, including the histone deacetylase transcriptional corepressor mSin3A and the novel nuclear protein PAM14, and they are also known components of the Tip60/NuA4 complex via interactions with the MRG binding protein (MRGBP). We present here the crystal structure of a prototypic MRG domain from human MRG15 whose core consists of two orthogonal helix hairpins. Despite the lack of sequence similarity, the core structure has surprisingly striking homology to a DNA-interacting domain of the tyrosine site-specific recombinases XerD, lambda integrase, and Cre. Site-directed mutagenesis studies based on the X-ray structure and bioinformatics identified key residues involved in the binding of PAM14 and MRGBP.
Assuntos
Proliferação de Células , Senescência Celular/fisiologia , Proteínas de Ligação a DNA/química , Homologia Estrutural de Proteína , Fatores de Transcrição/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Histona Acetiltransferases , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
MORF4-related gene on chromosome 15 (MRG15) is a core component of the NuA4/Tip60 histone acetyltransferase complex that modifies chromatin structure. We here demonstrate that Mrg15 null and heterozygous mouse embryonic fibroblasts exhibit an impaired DNA-damage response post gamma irradiation, when compared to wild-type cells. Defects in DNA-repair and cell growth, and delayed recruitment of repair proteins to sites of damage were observed. Formation of phosphorylated H2AX and 53BP1 foci was delayed in Mrg15 mutant versus wild-type cells following irradiation. These data implicate a novel role for MRG15 in DNA-damage repair in mammalian cells.
Assuntos
Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Reparo do DNA , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/efeitos da radiação , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Transativadores/deficiência , Transativadores/genética , Acetilação , Animais , Apoptose/efeitos da radiação , Núcleo Celular/metabolismo , Proliferação de Células/efeitos da radiação , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Raios gama/efeitos adversos , Deleção de Genes , Heterozigoto , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Enhancing factor (EF), a growth factor modulator, is the mouse homologue of human secretory group II phospholipase A(2). EF exhibits growth-promoting activity in vitro, in the presence of epidermal growth factor, and also brings about phenotypic transformation of normal cells. In order to ascertain the role of EF in vivo, a human keratin-14 promoter was used to drive the expression of EF ectopically to squamous epithelial cells. The founder mouse and its progeny showed abnormal whiskers and a scaly, beaded tail. In these mice, keratinization pattern of the epidermis was disturbed and parakeratosis and acanthosis were noted. The transgenic mice, TgK14-EF, expressed EF in the suprabasal layers of tail epidermis as well as in the epithelial cells of hair follicle and sebaceous glands of skin. Expression of EF along with hyperplasia was also observed in other squamous epithelia such as buccal mucosa, tongue and oesophagus. TgK14-EF mice homozygous for the transgene showed delayed and scanty hair growth although the mice were healthy and fertile. The hemizygous TgK14-EF mice were sensitive to a two-stage chemical carcinogenesis and developed a higher number of papillomas than their normal littermates over the course of the experiment. The conversion rate of papilloma to carcinoma was two fold higher in the transgenic mice.
Assuntos
Anormalidades Múltiplas/genética , Carcinoma de Células Escamosas/induzido quimicamente , Epiderme/anormalidades , Papiloma/induzido quimicamente , Fosfolipases A/fisiologia , Neoplasias Cutâneas/induzido quimicamente , Pele/enzimologia , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Carcinógenos/toxicidade , Carcinoma de Células Escamosas/genética , Fibrossarcoma/induzido quimicamente , Fibrossarcoma/genética , Predisposição Genética para Doença , Genótipo , Fosfolipases A2 do Grupo II , Humanos , Queratinas/genética , Camundongos , Camundongos Transgênicos , Neoplasia de Células Basais/induzido quimicamente , Neoplasia de Células Basais/genética , Papiloma/genética , Fenótipo , Fosfolipases A/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/fisiologia , Neoplasias Cutâneas/genética , Cauda/anormalidades , Cauda/patologia , Acetato de Tetradecanoilforbol/toxicidade , Transgenes , Vibrissas/anormalidadesRESUMO
Enhancing factor (EF) protein, an isoform of secretory phospholipase A2 (PLA2), was purified as a modulator of epidermal growth factor from the small intestine of the Balb/c mouse. It was for the first time that a growth modulatory property of sPLA2 was demonstrated. Deletion mutation analysis of EF cDNA carried out in our laboratory showed that enhancing activity and phospholipase activity are two separate activities that reside in the same molecule. In order to study the specific amino acids involved in each of these activities, two site-directed mutants of EF were made and expressed in vitro. Comparison of enhancing activity as well as phospholipase A2 activity of these mutant proteins with that of wild type protein helped in identification of some of the residues important for both the activities