RESUMO
Overexpression of the cytoplasmic oncoprotein Bcl2 blocks programmed cell death (apoptosis) in many cellular systems. To map the sequences in Bcl2 that are necessary for its activity, we created a library of deletion-scanning mutants of this 239-amino-acid protein and tested their abilities to block staurosporine-induced fibroblast apoptosis, using a novel transient-transfection assay. Phenotypes of informative mutants were then confirmed by assaying for inhibition of steroid-induced apoptosis in stably transfected T-lymphoid cells. In accordance with earlier results, we found that Bcl2 activity was only partially reduced after deletion of the hydrophobic tail that normally anchors it in cytoplasmic membranes. Essential sequences were found in the remainder of the protein and appeared to be organized in at least two discrete functional domains. The larger, more C-terminal region (within residues 90 to 203) encompassed, but extended beyond, two oligopeptide motifs called BH1 and BH2, which are known to mediate dimerization of Bcl2 and related proteins. The second, more N-terminal regions (within residues 6 to 31) was not required for protein dimerization in vivo, but its deletion imparted a dominant negative phenotype, yielding mutants that promoted rather than inhibited apoptotic death. Residues 30 to 91 were not absolutely required for function; by deleting most of this region along with the hydrophobic tail, we derived a 155-residue mini-Bcl2 that retains significant ability to inhibit apoptosis.
Assuntos
Apoptose/genética , Proteínas Proto-Oncogênicas/genética , Linhagem Celular , Fibroblastos/metabolismo , Fibroblastos/patologia , Técnicas de Transferência de Genes , Humanos , Mutação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Análise de SequênciaRESUMO
The human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex transactivators are posttranscriptional regulatory proteins that promote retroviral gene expression by interacting with specific viral mRNAs. Rev and Rex have markedly dissimilar amino acid sequences and RNA target specificities but are thought to act through the same cellular pathway. In this report, we demonstrate that short peptide domains which are required for effector activity in Rev and Rex are functionally interchangeable. Activity of these effector domains depends upon a previously unrecognized tetrapeptide motif that is present in both Rev and Rex and also in analogous proteins from other complex retroviruses. The conserved effector motif may mediate essential interactions of Rev, Rex, and other transactivators of this type with a common cellular cofactor.
Assuntos
Produtos do Gene rev/genética , Produtos do Gene rex/genética , Genes pX , Genes rev , HIV-1/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Produtos do Gene rev/análise , Produtos do Gene rex/análise , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Plasmídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/análise , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transfecção , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
The greater immune reactivity of females has been attributed in part to the influence of sex steroid hormones, but the underlying mechanisms are unknown. Here we report evidence that expression of the IFN-gamma gene may be subject to direct hormonal control. In a transient expression assay, the sex steroid 17 beta-estradiol markedly increases activity of the IFN-gamma promoter in lymphoid cells that express the appropriate hormone receptor. This effect is mediated by sequences in the 5'-flanking region of the gene, and can augment the effect of T cell-activating agents. Short term exposure to estradiol also increases IFN-gamma mRNA expression in Con A-treated murine spleen cells. Hormonal regulation of this pleiotropic cytokine may account in part for the ability of estrogen to potentiate many types of immune responses, and for the disproportionate susceptibility of females to autoimmune disease.
Assuntos
Estrogênios/farmacologia , Interferon gama/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/análise , Cloranfenicol O-Acetiltransferase/genética , Masculino , Camundongos , Dados de Sequência Molecular , Subpopulações de Linfócitos T/metabolismoRESUMO
Rev protein regulates nuclear export of viral mRNAs that contain a 240-base RNA sequence termed the Rev-response element (RRE). We demonstrate that an 88-base truncated RRE encompassing a known Rev binding site can mediate Rev responsiveness in vivo. Two tandem copies of this mutant function as efficiently as the full-length RRE.