RESUMO
Rat peroxisome assembly factor-2 (PAF-2) cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP92, using transient transfection assay. This cDNA encodes a 978-amino acid protein with two putative ATP-binding sites. PAF-2 is a member of a putative ATPase family, including two yeast gene products essential for peroxisome assembly. A stable transformant of ZP92 with the cDNA was morphologically and biochemically restored for peroxisome biogenesis. Fibroblasts derived from patients deficient in peroxisome biogenesis (complementation group C) were also complemented with PAF-2 cDNA, indicating that PAF-2 is a strong candidate for the pathogenic gene of group C peroxisome deficiency.
Assuntos
Adenosina Trifosfatases/genética , Teste de Complementação Genética , Microcorpos/enzimologia , ATPases Associadas a Diversas Atividades Celulares , Acil-CoA Oxidase , Aciltransferases/metabolismo , Adenosina Trifosfatases/análise , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Catalase/análise , Clonagem Molecular/métodos , Cricetinae , Citosol/enzimologia , DNA Complementar/genética , Fibroblastos , Humanos , Fígado/química , Dados de Sequência Molecular , Mutação , Oxirredutases/análise , Transtornos Peroxissômicos/genética , Transtornos Peroxissômicos/metabolismo , RNA Mensageiro/análise , Ratos , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
A protein named nuclear receptor binding factor-2 (NRBF-2) was identified by yeast two-hybrid screening, as an interaction partner of peroxisome proliferator-activated receptor alpha as well as several other nuclear receptors. NRBF-2 exhibited a gene activation function, when tethered to a heterologous DNA binding domain, in both mammalian cells and yeast.
Assuntos
Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transativadores/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Regulação da Expressão Gênica , Fígado/metabolismo , Dados de Sequência Molecular , Plasmídeos , Ratos , Alinhamento de Sequência , Transativadores/química , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Células Tumorais CultivadasRESUMO
The yeast two-hybrid screening was applied to cloning cDNAs of proteins that interact with peroxisome proliferator-activated receptor alpha (PPAR alpha). We obtained from a rat liver cDNA library a clone encoding a protein related to the ligand-binding domain of the members of nuclear hormone receptor superfamily, whereas apparently lacking the zinc-finger DNA-binding domain. This protein interacted with the activated forms of several nuclear receptors, and thus is a novel type of heterodimer-forming nuclear receptor.
Assuntos
Proteínas de Ligação a DNA/genética , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos , Ratos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Dedos de ZincoRESUMO
To identify the proteins which may modulate the functions of peroxisome proliferator-activated receptor (PPAR), a rat liver cDNA library was screened by a yeast two-hybrid system, using the mouse PPARalpha as a bait. A protein named nuclear receptor binding factor-1 (NRBF-1) was identified, which interacts not only with PPARalpha, but also with various nuclear hormone receptors in the presence of the respective ligands. Both the hinge and ligand-binding domains of PPARalpha are required for the interaction. NRBF-1 seems to be translocated to the nucleus by a piggyback mechanism, together with PPARalpha. NRBF-1 has a significant homology to the yeast protein MRF1, a putative transcription factor regulating the expression of mitochondrial respiratory proteins. NRBF-1 might be another type of nuclear receptor co-operator.